The polycomb group protein PCGF6 mediates germline gene silencing by recruiting histone-modifying proteins to target gene promoters

Abstract

Polycomb group (PcG) proteins are essential for maintenance of lineage fidelity by coordinating developmental gene expression programs. Polycomb group ring finger 6 (PCGF6) has been previously reported to repress expression of lineage-specific genes, especially germ cell-related genes in mouse embryonic stem cells (ESCs) via the noncanonical polycomb repressive complex PRC1.6. However, the molecular mechanism of this repression remains largely unknown. Here, using RNA-Seq, real-time RT-PCR, immunohistochemistry, immunoprecipitation, and ChIP analyses, we demonstrate that PCGF6 plays an essential role in embryonic development, indicated by the partially penetrant embryonic lethality in homozygous PCGF6 (Pcgf6^-/-)-deficient mice. We also found that surviving Pcgf6-deficient mice exhibit reduced fertility. Using the Pcgf6-deficient mice, we observed that ablation of Pcgf6 in somatic tissues robustly derepresses germ cell-related genes. We further provide evidence that these genes are direct targets of PCGF6 in ESCs and that endogenous PCGF6 co-localizes with the histone-modifying proteins G9A histone methyltransferase (G9A)/G9a-like protein (GLP) and histone deacetylase 1/2 (HDAC1/2) on the promoters of the germ cell-related genes. Moreover, the binding of these proteins to their target genes correlated with methylation of Lys-9 of histone 3 and with the status of histone acetylation at these genes. Moreover, the recruitment of G9A/GLP and HDAC1/2 to target promoters depended on the binding of PCGF6. Our findings indicate that PCGF6 has a critical role in safeguarding lineage decisions and in preventing aberrant expression of germ cell-related genes.

Keywords: G9A histone methyltransferase; G9a-like protein 1 (Glp); G9a/Glp; PRC1.6; Pcgf6; embryonic stem cell; embryonic stem cells; epigenetic regulation; gene knockout; gene regulation; germline gene silencing; histone deacetylase (HDAC); histone deacetylase 1/2 (Hdac1/2); histone methylation; knockout mice; polycomb; polycomb group ring finger 6 (Pcgf6).

Figure 1.

Pcgf6 null mice exhibit partially penetrant embryonic lethality. A, schematic of the gene targeting strategy used to generate Pcgf6 mutant mice. The WT Pcgf6 locus is shown at the top. The targeting vector contains loxP sites flanking exons 2–5 and a neomycin gene flanked by FRT sites. F, R1, R2 are primers used to determine genotypes. B, genomic PCR for analysis of Pcgf6 gene status. The primers F, R1, and R2 shown in A and DNA from toes of mice were used to determine genotypes. C, Western blotting demonstrating changes in the protein levels of the indicated genes in the liver, brain, and testis of indicated genotypes. Protein loading is 40 μg. Gapdh was used as a control. D, genotype distribution of progeny of different intercrosses. ***, p < 0.005 (χ² test), indicating that the probability of conforming to the Mendelian law is <0.5%. E, body size of the Pcgf6^+/+ and Pcgf6^–/– mice at 6 weeks. F, body weight of Pcgf6^−/− mice from birth to adulthood compared with WT mice. n = 6. G, fecundity of Pcgf6^+/+, Pcgf6^+/–, and Pcgf6^–/– mice. n, number of mice. a, p < 0.01(t test) versus +/+ × +/+.

Figure 2.

Pcgf6-deficient mice dramatically reduced in fertility. A, representative image of the testes and epididymides of the WT and Pcgf6^−/− mice. Scale bars, 2 mm. B, testis and epididymis as a percentage of body weight of mice with the indicated genotype. C, H&E staining of representative sections of the testes and epididymis of the WT and Pcgf6^−/− mice. Scale bars, 50 μm (cauda epididymis) or 20 μm (others). D, normal and abnormal sperms in WT and Pcgf6^−/− mice at 8 weeks of age. Red arrows, defective sperms. E and F, the proportions of normal sperm and the sperm motility of mice with the indicated genotype at 8 weeks of age. Data are represented as the means ± S.D. (error bars) from five mice for each genotype. Sperm motility was measured using a CASA CEROS version 12 sperm analysis system (Hamilton Thorne). PR, progressively motile; NP, nonprogressively motile; IM, immotile. G, volcano plot of differentially expressed genes in testis between WT and Pcgf6^−/− mice. H, GO analyses showing the biological functions of down-regulated genes in Pcgf6^−/− testis. I, the log₂(-fold change) of the several down-regulated genes related to sperm function in Pcgf6^−/− testis.

Figure 3.

Pcgf6 represses the expression of germline-related genes with binding sites of Max and E2f6 in the liver and brain. A, RNA-Seq heat map of transcripts with 2-fold expression differences and p < 0.05 in liver and brain comparing WT mice with Pcgf6^−/− mice. B, GO analysis of biological functions of 207 up-regulated genes in the liver and brain. C, Venn diagrams of up-regulated target genes and germline-related genes in the liver and brain of Pcgf6^−/− mice. D, log₂(-fold changes (KO versus WT)) in the expression of the 11 germline-related genes up-regulated in both liver and brain. E, motif analysis of 41 germline-related genes in up-regulated genes of liver and brain. F, the overlap between E2f6-binding sites containing genes and Max-binding sites containing genes.

Figure 4.

Pcgf6 represses the expression of germline-related genes almost in all somatic organs. Relative expression of the indicated germline-related genes in various tissues of Pcgf6^−/− mice determined by qRT-PCR. Actin was used as a control for normalization. Hprt1 was used as a negative control. Data are from three biological replicates each with three technical replicates. Error bars, S.D. n.s., nonsignificant (two-tailed Student's t test).

Figure 5.

G9a/Glp and Hdac1/2 repress the expression of Pcgf6 targeted germline genes. A, IP–Western blot analysis showing physical interaction of Pcgf6, Hdac1/2, and G9a/Glp in cell extracts from WT and Pcgf6^−/− ESCs. B, Western blot analysis showing the methylation levels of H3K9 in G9a^−/−, Glp^−/−, and G9a/Glp^−/− ESCs. C, Western blot analysis showing the acetylation levels of H3 in Hdac1^−/− and Hdac2^−/− ESCs. D, the mRNA levels of germline-related genes in ESCs of the indicated genotype or in ESCs treated with the indicated inhibitors. All cells were treated with inhibitors for 72 h. DMSO is the solvent control. Error bars, S.D. of three independent experiments. The asterisks arranged vertically refer to significant differences compared with WT ESCs. Horizontally arranged asterisks indicate significant differences between groups.

Figure 6.

Pcgf6 plays a key role in recruiting G9a/Glp and Hdac1/2 to germline-related genes. A, mRNA expression analysis of the selected genes in ESCs of the indicated genotype. B–D, ChIP-qPCR analysis showing enrichment of the indicated proteins on the selected gene promoters in WT and Pcgf6^−/− ESCs. Error bars, S.D. of three independent repeats. E, a schematic model showing that recruiting Hdac1/2 and G9a/Glp is an important mechanism used by Pcgf6 to modify histone tails and repress the expression of germline-related genes (see “Discussion” for a detailed description).